In a conventional multiplexed reverse dot blot hybridization assay protocol used in the process of detecting the presence of particular alleles in a sample, selected loci in the double-stranded genomic DNA are first amplified using pairs of forward and reverse primers, and one designated strand of each of the double-stranded amplicons is removed, for example by enzymatic digestion or magnetic separation. Only the remaining strands, preferably labeled, are placed in contact with a set of cognate probes, spotted or otherwise placed on a substrate, such as a strip of nitrocellulose, or displayed on encoded microparticles in preparation for a hybridization assay. Hybridization is typically detected based on the presence of label associated with the set of captured targets or with the corresponding probes. Decoding allows determination of the subsequences of the strands captured by particular probes, indicating that the capturing probes are complementary to such subsequences.
Removal of the designated strands is intended to improve the efficiency of capture of the remaining strands to probes, by eliminating strand-strand re-annealing, a process which competes with annealing to cognate capture probes, and would otherwise take place without strand selection and removal in the protocol.
Strands can be removed by digestion, wherein strands selected for removal are first phosphorylated, and then enzymatically digested using a digestion enzyme such as λ-endonuclease for the phosphorylated strands. Strands can also be removed by magnetic separation. Both digestion as well as magnetic separation add cost and labor to the assay protocol. A preferable alternative would be to generate single-stranded fragments from amplicons, thereby eliminating the need for digestion or magnetic separation.
Several methods are known to generate single-stranded fragments of random length from double-stranded DNA. In the conventional Maxam and Gilbert sequencing method (A. Maxam and W. Gilbert, PNAS 74, p. 560, 1977), fragments of double-stranded DNA are generated by selective chemical degradation of multiple copies of the DNA species to be sequenced. Conditions are adjusted to produce fragments of all possible lengths; that is, fragments can be separated into fractions differing from one another in length by only a single base. When ordered, the sequence of fractions with their respective terminal bases represents the sequence of the original DNA species.
E. Southern et al. have also generated random-sized single-stranded fragments from double-stranded DNA to facilitate the transfer of DNA from agarose gels for blotting on membranes for further analysis by hybridization with oligonucleotide probes, in what represents a dot blot format. Conditions are adjusted to produce fragments that are sufficiently short to minimize entanglement within the gel.
To date, no one has suggested the use of amplicon fragments without strand digestion or separation for use in reverse dot blot hybridization assay formats. Thus, there is no suggestion that, for multiplexed analysis of polymorphisms (MAP), the use of a complex mixture of highly heterogeneous target fragments is preferable to the use of a single, or at most a few, target sequences. Further, to use fragmentation for MAP, unless conditions are adjusted correctly, some or all polymorphic sites may be eliminated, or labeling may be impractical, and none of these problems or their solutions have been suggested.
One conventional method of labeling amplicons is to perform the amplification with 5′-terminally labeled primers so as to produce end-labeled amplicons. This end-labeling method requires that labeled strands remain intact because, after fragmentation, none but a small portion of the fragments containing the 5-terminus will be labeled. Therefore, a different method of labeling is needed where the amplicons are to be fragmented.